Spore-containing probiotic compositions and methods

ABSTRACT

A method for preparing a shelf stable spore-containing probiotic alimentary additive is disclosed, the method including providing a liquid slurry including spores from at least one spore-forming probiotic bacterial strain, a saccharide, and a humectant; and pasteurizing the liquid slurry to yield the alimentary additive. The alimentary additive may have a water activity (Aw) of less than about 0.7. The alimentary additive may have a pH from about 2.0 to 9.5. A plurality of the spores may remain in an un-germinated state and uniformly suspended for at least two months after the pasteurizing to yield a shelf stable spore-containing probiotic alimentary additive.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/837,589 filed Apr. 23, 2019, the entirecontents of which is hereby incorporated by reference in its entirety.

FIELD

Spore-containing probiotic compositions and methods are provided.

BACKGROUND

Probiotic microorganisms when consumed in adequate amounts can confer ahealth effect on the host. Probiotics today find applications in diversefields such as food, meal replacements, dietary supplements,nutraceuticals, over the counter drugs as well as prescription drugs.Probiotic products include dairy products and probiotic-fortified food.The products generally require storage at low temperatures to maintainthe probiotic activity. Growth of probiotic microorganisms in theseproducts can give rise to offensive odor to the products.

Many probiotic formulations are provided as solid oral dosage forms.While liquid formulations can facilitate ease of application in food andbeverage processing applications, to date such formulations do not existdue to the drawbacks associated with liquid probiotic formulations,e.g., achieving a uniform suspension to enable dosage accuracy whilemaintaining the shelf-life stability of the formulation. Liquidprobiotic formulations typically include sugars that can act assubstrates and can enhance the risk of undesired metabolite production.Gas producing probiotics can result in bulging of the containers andproduction of offensive odors and taste. Furthermore, metaboliteproduction can result in death of the probiotic strain and lower viablecounts of the probiotic. As a result, such liquid probiotic formulationsneed to be stored in cold conditions (e.g., 2° C. to 8° C.), which canlimit their use in food applications and increase distribution costs.

There are several commercially available liquid probiotic formulationsthat are available as Health Supplements. For example, “Enterogermina”produced and marketed by Sanofi is a liquid oral suspension thatcontains 4 strains of Bacillus clausii, a species distinct from Bacilluscoagulans, in purified water at a concentration of 2 billion CFU/5 mL.Enterogermina is specifically recommended for use by people withdiarrhea. “Bacilliq SC” is also produced and marketed by Synergia LifeSciences Pvt. Ltd. and does not require cold chain storage. Bacilliq SCcontains distinct probiotics strains, Bacillus subtilis HU58 andBacillus coagulans SC208, and sugar.

In addition, a conventional probiotic composition is disclosed in U.S.Patent Application Publication No. 2015/0118203 by NCH Corporation (“the'203 publication”). The '203 publication discloses a system fordelivering probiotic compositions by gravity feed or non-contact pump toa point of consumption by a plant or animal. The probiotic compositionscomprise one or more species of bacteria in spore form, a thickener, oneor more acids or salts of acids and optionally a water activity reducer.

There is no consideration in the '203 publication for human consumptionof its probiotic composition. Further, the '203 publication hasdisadvantages in repeatability, formulation and lack of food safetyprocessing with regards to addition to foods intended for humanconsumption. For example, the '203 publication does not describe anyprocessing information with regard to repeatable production of theproduct. This could lead, for example, to poorly dispersed, undissolvedor not hydrated ingredients, which in turn, can lead to a formulationthat is not suitable for prevention of microbial growth orcontamination. Further, lack of a 5-log kill step, for example, presentsinherent risk to not only the probiotic composition of the '203publication, but also to potential foods being dosed with thecomposition. Without a properly defined 5-log kill step, risk ofmicrobial contamination, which may show dormancy in the inventionscomposition, will grow once added to food products that present optimalgrowth factors including water and food source. Having no defined killstep also presents the opportunity for background contamination throughuse of ingredients including corn syrup, prebiotics, and thickeners.

In light of the technical issues summarized above, one of the goals ofthe present disclosure was to provide an alimentary probioticformulation and a processing method that is reproducible by those ofordinary skill in the art and avoids the disadvantages of the prior artand conventional compositions and methods, and guarantees a product thatis safe and effective for human consumption.

SUMMARY OF THE DISCLOSURE

The disclosure provides for an improved delivery of spore-containingprobiotic compositions that can retain their cellular viability underambient, refrigerated, and frozen storage conditions over extended timeperiods and that can then be added to and remain stable in food andbeverage products.

Accordingly, in certain embodiments, a method for preparing a shelfstable spore-containing probiotic alimentary additive, comprisesproviding a liquid slurry comprising spores from at least onespore-forming probiotic bacterial strain, a saccharide, and a humectant;and pasteurizing the liquid slurry to yield the alimentary additive,wherein the alimentary additive comprises a water activity (Aw) of lessthan about 0.7; the alimentary additive comprises a pH from about 2.0 to9.5; and a plurality of the spores remain in an un-germinated state andremain uniformly suspended for at least two months after thepasteurizing step to yield a shelf stable spore-containing probioticalimentary additive.

In certain embodiments, at least 90, 95, or 99% of the spores remain inan un-germinated state for at least two months after the pasteurizingstep. In certain embodiments, at least 90, 95, 99% of the spores remaingermination competent. In certain embodiments, at least 90, 95, 99% ofthe spores germinate following exposure to a germination-permissivecondition.

In certain embodiments, at least 90, 95, or 99% of the spores germinatefollowing ingestion by a mammal. In certain embodiments, thegermination-permissive condition comprises at least 1 Aw and 37° C. Incertain embodiments, at least 90% of the spores are viable afterpasteurization.

In certain embodiments, the alimentary additive comprises less than 1%(w/v) of vegetative bacterial cells.

In certain embodiments, the at least one spore-forming probioticbacterial strain comprises at least one Bacillus bacterial strain. Incertain embodiments, the at least one spore-forming probiotic bacterialstrain comprises at least one of Bacillus coagulans bacterial strain, atleast one of Bacillus subtilis bacterial strain, at least one ofBacillus clausii bacterial strain, at least one of Bacillus indicusbacterial strain, at least one of Bacillus licheniformis bacterialstrain, at least one of Bacillus pumilus bacterial strain, at least oneof Bacillus amyloliquifaciens bacterial strain, or at least one ofBacillus megaterium bacterial strain. In certain embodiments, the atleast one Bacillus coagulans bacterial strain comprises Bacilluscoagulans GBI-30 strain (ATCC Designation Number PTA-6086).

In certain embodiments, a plurality of spores is present in thealimentary additive in an amount from about 500 million CFU/g to 1trillion CFU/g by weight of the composition.

In certain embodiments, the at least one humectant is at least one ofglycerin, a sugar, a salt, propylene glycol, or a gum.

In certain embodiments, the alimentary additive further comprises atleast one rheology modifier that does not contain inulin, wherein the atleast one rheology modifier is present in an amount from about 0.01% to0.3% by weight of the composition. In certain embodiments, the at leastone rheology modifier is at least one of xanthan gum, gum arabic, gellangum, guar gum, or carrageenan.

The disclosure also provides for a spore-containing probioticcomposition. In certain embodiments, a spore-containing probioticcomposition comprises a liquid slurry comprising, a probiotic materialcomprising a plurality of spores from at least one spore-formingprobiotic bacterial strain wherein at least 90% of the spores are viableat pasteurization times and temperatures; a saccharide, and at least onehumectant, or combinations thereof, wherein the composition has a wateractivity (Aw) of less than about 0.7 and a pH of less than about 5.0.

The disclosure also provides for a food product, comprising thespore-containing probiotic composition.

The disclosure also provides a beverage product, comprising thespore-containing probiotic composition.

DETAILED DESCRIPTION

The compositions and methods described herein provide a solution to thedrawbacks and limitations of existing probiotic products.Spore-containing (non-vegetative) probiotic compositions are providedthat can survive specific time-temperature, pH, and water activityenvironments when pasteurized to deliver consistent microbiologicalviability, and subsequent activity when added to food and beverageproducts. For example, the probiotic viability of the spore-containingprobiotic compositions can be from about 500 million (M) colony formingunit (CFU)/gram (g) to 1 trillion (T) colony forming unit (CFU)/gram (g)by weight of the composition after the pasteurization process.

The spore-containing probiotic compositions described herein can be inthe form of a uniformly suspended and dispersible liquid slurry. Theliquid slurry is designed such that it can remain stable in ambientstorage conditions at least before being added to a food or beverageproduct (e.g., a refrigerated food or beverage product or a frozen foodor beverage product or ambient food or ambient beverage). The liquidslurry form can be more easily and economically added to a food orbeverage product as compared to a powdered form, e.g., lower processingtime, less expensive to produce, manufacture, and process, and uniformlyconcentrated throughout the composition. As used herein, “food” refersto human food, pet food (e.g., food designed for consumption bycompanion animals such as dogs, cats, and the like), or animal feed(e.g., food designed for consumption by non-companion animals such aslivestock and the like). As used herein, “beverage” refers a humanbeverage, a pet beverage (e.g., a beverage designed for consumption bycompanion animals such as dogs, cats, and the like), or animal beverage(e.g., a beverage designed for consumption by non-companion animals suchas livestock and the like). Further, the liquid slurry is designed towithstand pasteurization methods, such as High Temperature Short Time(HTST), High Pressure Pasteurization (HPP), and the like. In someembodiments, the spore-containing probiotic compositions can have anextended shelf-life. For example, the extended shelf-life can be up tothree months, up to six months, or up to the shelf-life of the food orbeverage product. Further, the compositional makeup of the liquid slurryresults in viscosity and rheological properties that allow the liquidslurry to be added to a food or beverage product using any suitableliquid delivery system (e.g., dosage systems).

The spore-containing probiotic compositions generally include aprobiotic material. In some embodiments, the probiotic material presentin the composition can be in an amount from about 5% to 70%, from about30% to 70%, from about 40% to 60%, from about 40% to 50%, from about 5%to 30%, from about 5% to 25%, or from about 5% to 20% by weight of thecomposition. In other embodiments, the probiotic material can be presentin the composition in an amount between any of these recited values.

The probiotic material includes a plurality of spores and at least oneexcipient as described below. The plurality of spores are from at leastone spore-forming probiotic bacterial strain. In some embodiments, theplurality of spores present in the composition can be in an amount fromabout 500 million (M) CFU/g to 1 trillion (T) CFU/g, about 1 billion (B)CFU/g to 1 trillion (T) CFU/g, from about 500 million (M) CFU/g to 5billion (B) CFU/g, or from about 500 million (M) CFU/g to 10 billion (B)CFU/g by weight of the composition. In other embodiments, the pluralityof spores present in the composition can be in an amount between any ofthese recited values.

The at least one spore-forming bacterial strain can include anyspore-forming bacterial strain(s). In some embodiments, the at least onespore-forming probiotic bacterial strain can be at least one Bacillusbacterial strain. Non-limiting examples of suitable spore-formingbacterial strains can include at least one of Bacillus coagulansbacterial strain, at least one of Bacillus subtilis bacterial strain, atleast one of Bacillus clausii bacterial strain, at least one of Bacillusindicus bacterial strain, at least one of Bacillus licheniformisbacterial strain, at least one of Bacillus pumilus bacterial strain, atleast one of Bacillus amyloliquifaciens bacterial strain, at least oneof Bacillus megaterium bacterial strain, or any combinations thereof.

In some embodiments, the at least one spore-forming probiotic bacterialstrain can be at least one Bacillus coagulans bacterial strain. Forexample, the at least one Bacillus coagulans bacterial strain caninclude Bacillus coagulans GBI-30 strain (ATCC Designation NumberPTA-6086). Additional details of the Bacillus coagulans GBI-30 straincan be found in U.S. Pat. Nos. 6,849,256, 7,713,726, and 8,277,799, eachof which is incorporated herein by reference in its entirety.

The homogeneity of the spore distribution throughout thespore-containing probiotic compositions can be stable. For example, thecomposition does not undergo substantial sedimentation (e.g., greaterthan 5% of the amount of spores by volume of the spore-containingprobiotic compositions) or phase separation (e.g., greater than 5% ofthe amount of spores by volume of the spore-containing probioticcomposition). This homogeneity, alone or in combination with, thespecific gravity of the spore-containing probiotic composition canfacilitate reproducible dosing of the spore-containing probioticcomposition into food or beverage products. In some embodiments, thespecific gravity of the composition can be from about 1.0 to 1.5 or fromabout 1.25 to 1.45. In other embodiments, the specific gravity of thecomposition can be between any of these recited values.

In some embodiments, the probiotic material can also include at leastone saccharide. Nonlimiting examples of suitable saccharides includemonosaccharides and polysaccharides, such as inulin or maltodextrin,galactoosaccharides, fructooligosaccharides. For example, the inulin canbe present in an amount from about 75% to 95%, from about 80% to 90%, orfrom about 75% to 80% by weight of the probiotic material. In suchembodiments, the inulin can function as a rheology modifier.

In other embodiments, the probiotic material does not include inulin. Insuch embodiments, inulin can be separately added to the spore-containingprobiotic composition. As used herein, “added inulin” is used to referto inulin that is not contained within the probiotic material, butrather separately added to the to the spore-containing probioticcomposition. For example, in some embodiments, separately added inulincan be present in an amount from about 0.1% to 30%, from about 0.1% to25%, or from about 0.1% to 20% by weight of the composition. In suchinstances, the separately added inulin can function as a rheologymodifier.

In some embodiments, the probiotic material can include maltodextrin. Insuch embodiments, inulin can be separately added, referred to herein as“added inulin,” to the spore-containing probiotic composition. Forexample, in some embodiments, added inulin can be present in an amountfrom about 0.1% to 30%, from about 0.1% to 25%, or from about 0.1% to20% by weight of the composition.

In some embodiments, the probiotic material can include non-fat drymilk. In such embodiments, inulin can be separately added, referred toherein as “added inulin,” to the spore-containing probiotic composition.For example, in some embodiments, separately added inulin can be presentin an amount from about 0% to 30%, from about 0% to 25%, or from about0% to 20% by weight of the composition.

In addition to probiotic material, the spore-containing probioticcompositions can include water. In some embodiments, water is present inan amount from about 20% to 60%, from about 20% to 50%, or from about20% to 40% by weight of the composition.

Further, the dormancy of the spores of the spore-containing probioticcompositions can be maintained by minimizing the water activity, e.g.,to less than about 0.95 water activity (Aw). That is, low water activitycan inhibit germination of the spores. Low water activity also reducessedimentation or phase separation within the compositions for a periodof time, e.g., for about up to three months, up to six months, or up tothe shelf-life of a food or beverage product containing thecomposition(s). As a result, the spore-containing probiotic compositionscan remain uniformly suspended, prior to adding the compositions to aproduct, e.g., a food or beverage product. That is, unlike conventionalspore-containing probiotic compositions, e.g., Enterogermina that isproduced and marketed by Sanofi, the present spore-containing probioticcompositions can be added to a food product or beverage product withoutthe need to thoroughly mix the compositions prior to addition to thefood or beverage product. Thus, an advantage of the spore-containingprobiotic compositions described herein is maintenance of uniformsuspension for extended periods of time.

Moreover, the inhibition of spore germination via low water activity,can lead to other advantages. For example, prior to desired use, thespores can remain uniformly dispersed throughout the compositions forextended periods of time, and thereby increasing shelf-life. Further,the spores of the spore-containing probiotic compositions can remain intheir resistant states such that they do not germinate or proliferate inresponse to environmental factors. As a result, the spores can survivepost-pasteurization, such as High Temperature Short Time (HTST), HighPressure Pasteurization (HPP), and the like.

In some embodiments, the water activity of the spore-containingprobiotic compositions can be from about 0.001 Aw to about 0.99 Aw. Insome embodiments the water activity of the spore-containing probioticcompositions is less than or equal to about 0.95 Aw, or less than orequal to about 0.90 Aw, or less than or equal to about 0.85 Aw, or lessthan or equal to about 0.80 Aw, or less than or equal to about 0.75 Aw,or less than or equal to about 0.70 Aw, or less than or equal to about0.65 Aw, or less than or equal to about 0.60 Aw, or less than or equalto about 0.55 Aw, or less than or equal to about 0.50 Aw, or less thanor equal to about 0.45 Aw, or less than or equal to about 0.40 Aw, orless than or equal to about 0.35 Aw, or less than or equal to about 0.30Aw, or less than or equal to about 0.25 Aw, or less than or equal toabout 0.20 Aw, or less than or equal to about 0.15 Aw, or less than orequal to about 0.10 Aw, or less than or equal to about 0.05 Aw, or lessthan or equal to about 0.01 Aw, or less than or equal to about 0.005 Aw,or less than or equal to about 0.004 Aw, or less than or equal to about0.003 Aw, or less than or equal to about 0.002 Aw. This parameter can beachieved by the inclusion of at least one humectant. For example, insome embodiments, the spore-containing probiotic composition can includeat least one humectant. As used herein, a “humectant” is a hygroscopicsubstance, one that attracts and retains water. The at least onehumectant can be glycerin. The glycerin can be in an amount from about30% to 75%, from about 30% to 70%, from about 40% to 75%, from about 50%to 75%, from about 55% to 65%, from about 50% to 70%, from about 40% to60%, or from about 40% to 50% by weight of the composition. Non-limitingexamples of other suitable humectants can include sugars, e.g., sorbitol(e.g., from about 30% to 75% by weight of the composition), salts (e.g.,from about 30% to 75% by weight of the composition), propylene glycol(e.g., from about 30% to 75% by weight of the composition), and gums,e.g., xanthan acacia gum (e.g., from about 10% to 30% by weight of thecomposition).

The spore-containing probiotic compositions can include at least onerheology modifier that does not contain inulin and/or added inulin.Non-limiting examples of suitable rheology modifiers that do not containinulin and/or added inulin include xanthan gum, guar gum, gum arabic,carrageenan, gellan gum, and the like. In some embodiments, the at leastone rheology modifier that does not contain inulin and/or added inulinis present in an amount from about 0.01% to 0.3% by weight of thecomposition. For example, in some embodiments, the at least one rheologymodifier that does not contain inulin and/or added inulin is xanthan gumthat is present an amount from about 0.01% to 0.3% by weight of thecomposition. In other embodiments, the at least one rheology modifierthat does not contain inulin and/or added inulin is gum arabic that ispresent an amount from about 0.01% to 0.3% by weight of the composition.In other embodiments, the at least one rheology modifier that does notcontain inulin and/or added inulin is gellan gum that is present in anamount from about 0.01% to 0.3% by weight of the composition.

In some embodiments, the spore-containing probiotic compositions do notcontain salt. In other embodiments, the spore-containing probioticcomposition can contain salt that is present in an amount that is lessthan about 10%, less than about 5%, less than about 1%, less than about0.1%, less than about 0.01%, less than about 0.001%, or less than about0.0001% by weight of the composition. Non-limiting examples of saltinclude table salt, e.g. NaCl, kosher salt, iodized salt, sea salt,Himalayan salt. Other salts include, for example, chloride salts ofpotassium, magnesium, calcium, ammonium.

In some embodiments, the spore-containing probiotic compositions canhave a viscosity from about 25 centipoise (cP) to 2000 centipoise (cP)measured at a temperature of 4° C. For example, the viscosity of thespore-containing probiotic compositions can be from about 5 cP to100,000 cP, from about 5 cP to 10,000 cP, or from about 5 cP to 50,000cP measured at a temperature of 4° C.

The spore-containing probiotic compositions can also include additionalelements, such as natural flavor(s), preservatives, acidulants, etc.,and any combination thereof. For example, in some embodiments, thespore-containing probiotic compositions can include at least one naturalflavor that is present in an amount from about 0% to 2% by weight of thecomposition. In some embodiments, the spore-containing probioticcompositions can include at least one preservative that is present in anamount from about 0% to 2% by weight of the composition. As used herein,“preservative,” refers to an ingredient that inhibits the growth ofundesirable microorganisms. Non-limiting examples of suitablepreservatives include potassium sorbate, sodium benzoate, sorbic acid,benzoic acid, and propionic acid.

In some embodiments, the spore-containing probiotic compositions caninclude at least one acidulant that is present in an amount from about0% to 2% by weight of the composition. As used herein, “acidulant,”refers to an ingredient that lowers the pH of a composition to a finalpH (e.g., below about 5). The at least one acidulant can be an organicor mineral acid. Non-limiting examples of suitable acidulants includelactic acid, acetic acid, citric acid, hydrochloric acid, malic acid,and phosphoric acid. The addition of at least one acidulant to thespore-containing probiotic compositions can modify the pH of thecompositions so as to, for example, inhibit the growth of spoilagemicroorganisms. In some embodiments, the pH of the spore-containingprobiotic compositions that include an acidulant (e.g., lactic acid),can have a pH of less than 4.6. For example, in some embodiments, thespore-containing probiotic compositions can have a pH from about 2.0 to5, from about 2.5 to 5, or from about 3.0 to 5.

In some embodiments, the spore-containing probiotic compositions canhave a pH from about 2.0 to 9.5, from about 3.0 to 8.0, or from about2.5 to 8.5.

The spore-containing probiotic compositions can be produced using anysuitable method. For example, in some embodiments, the method caninclude mixing water and glycerin together to form a first mixture,adding at least one rheology modifier to the probiotic material (e.g.,at a 1:10 ratio) to form a second mixture, and then combining and mixing(e.g., under high shear) the first and second mixtures to form a liquidslurry. The method can also include adding at least one acidulant, e.g.,lactic acid, or preservative, e.g., potassium sorbate, sodium benzoate,and/or sorbic acid. The method can also include, after the addition ofat least one acidulant or preservative, homogenizing the liquid slurryunder high shear. After homogenization, the liquid slurry can bepasteurized (e.g., via HPP or HTST), thereby forming a spore-containingprobiotic composition and thereafter packaged (e.g., in a 0.1-20 L bagwith or without a cap). Alternatively, after the addition of at leastone acidulant or preservative, the step of homogenizing the liquidslurry under high shear, thereby produces the spore-containing probioticcomposition, which can then be packaged. In some embodiments, thepackaged the spore-containing probiotic composition can then bepasteurized (e.g., via HPP).

The packaged spore-containing probiotic composition can be added, forexample, to a food or beverage product, e.g., via mixing, e.g., low orhigh agitation, or spraying. In some embodiment, the delivery of thepackaged slurry into a food or beverage product can be carried under outunder aseptic conditions.

A spore-containing probiotic liquid slurry composition can be anadditive to a food or beverage product. The food or beverage product canbe refrigerated, frozen, or ambient.

The spore-containing compositions and methods may be further understoodwith the following non-limiting examples.

EXAMPLES Example 1: Preparation of Spore-Containing ProbioticCompositions

Sixteen different samples of spore-containing probiotic compositionswere prepared using the exemplary procedure described below.

Water is heated or cooled in a jacketed stainless steel mixing vessel to40° F.-100° F. The water is agitated with a high shear dissolver speed20%-80% and side sweep agitation 50%-100%. During agitation, acidulantis added to the water until the pH is between 4.0-4.6. Optionally, apreservative is added. Xanthan gum and/or gum arabic and/or gellan gumis/are mixed with the spore-containing probiotic powder at approximatelya 1:10 ratio. Dry matter is added to the water/acidulant and blendedwith a high shear dissolver speed 20%-80% and side sweep agitation50%-100% to form a liquid slurry. This is mixed for 5 minutes. Duringagitation, glycerin at 40° F.-100° F. is added to the liquid slurry. Theliquid slurry is mixed for 5-20 minutes using a high shear dissolverspeed 20%-80% and side sweep agitation 50%-100%. The liquid slurry istransferred from the vessel and through inline high shear equipment(e.g., IKA, Greerco, GEA, Niro Soavi Homogenizer). The liquid slurry isthen transferred from the shear equipment into and through heattreatment equipment (e.g., plate or scraped surface heat exchanger) toheat the liquid slurry to a temperature between 160° F.-200° F. for15-45 seconds. The heated liquid slurry is then cooled to a temperatureof 50° F. or less via inline cooling heat exchangers (plate or scrapedsurface). The cooled liquid slurry is then packaged into a containersuch as a 0.1 L-20 L bag-in-box.

The compositional makeup of samples 1-8 and 9-16 are shown in Table 1Aand 1B, respectively.

TABLE 1A Compositional Makeup of Samples 1-8 SAMPLE SAMPLE SAMPLE SAMPLESAMPLE SAMPLE SAMPLE SAMPLE 1 2 3 4 5 6 7 8 Weight % Weight % Weight %Weight % Weight % Weight % Weight % Weight % Water 53.177 51.877 41.45439.500 27.500 29.450 29.400 Glycerin 50.000 89.500 50.000 55.000 55.000Probiotic 1.300 2.600 13.000 10.000 10.000 10.000 15.000 15.000 MaterialA (Spore-containing powder with 75-90% by wt. Inulin and 15 billionSpores) Gum Arabic 45.000 45.000 45.000 12.000 Gellan Gum 0.023 0.0230.046 0.050 0.100 Natural Flavor 0.500 0.500 0.500 0.500 0.500 0.5000.500 0.500 Total 100.000 100.000 100.000 100.000 100.000 100.000100.000 100.000

TABLE 1B Compositional Makeup of Samples 9-16 SAMPLE SAMPLE SAMPLESAMPLE SAMPLE SAMPLE SAMPLE SAMPLE 9 10 11 12 13 14 15 16 Weight %Weight % Weight % Weight % Weight % Weight % Weight % Weight % Water29.350 29.500 25.890 28.520 29.500 27.650 27.650 27.650 Glycerin 55.00054.455 43.000 50.200 54.465 46.000 46.000 46.000 Probiotic 15.000 15.00030.000 15.000 25.000 25.000 25.000 Material A (Spore-containing powderwith 75-90% by wt. Inulin and 15 billion Spores) Probiotic B 15.000(Maltodextrin and 75 billion spores) Xanthan Gum 0.045 0.010 0.080 0.0350.050 0.050 0.050 Gellan Gum 0.150 Inulin-added 5.000 Natural Flavor0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 Potassium Sorbate 0.100Sodium Benzoate 0.100 Sorbic Acid 0.100 Lactic Acid 0.500 0.600 0.7000.500 0.700 0.700 0.700 Total 100.000 100.000 100.000 100.000 100.000100.000 100.000 100.000

Example 2: Characterization of Spore-Containing Compositions

Samples 1-16 as prepared in Example 1 were each characterized. Table 2shows the various analyses performed and the methods used. Table 3Ashows the results of these analyses for Samples 1-8 and Table 3B showsthe results of these analyses for Samples 9-16.

TABLE 2 Analysis Performed Analysis Method ofDetermination/Instrumentation Water Activity A sample was transferredinto a disposable sample cup, completely covering the bottom of the cup,if possible. The disposable sample cup was then placed into an Aqua LabTuneable Diode Laser Water Activity Meter for analysis. The Aqua Labreading cycle continues until the rate of change of three consecutivereadings are less than 0.0005 A_(w) of each other. When the Aqua LabTuneable Diode Laser Water Activity Meter has finished its read cycle,the water activity and read is displayed. pH Thermo Scientific Orionseries benchtop pH probe was used to determine the pH of a sample.Sedimentation A 1000 mL of a sample was transferred into a 1000 mLplastic clear container and stored at a temperature of 19° C.-23° C.After 24 hours, the sample was visually assessed for sedimentation.Phase A 1000 mL of a sample was transferred in a Separation 1000 mLplastic clear container and stored at a temperature of 19° C.-23° C.After 24 hours, the sample was visually assessed for phase separation.Particle Size A sample was dispersed in dispersant and placed into aMalvern Mastersizer 3000 Laser Diffraction Instrument. Analysis wasperformed on a sample using Mastersizer software v.3.62. The analysiswas carried out under the following testing parameters: Dispersantproperties Dispersant name Water Refractive index    1.000 Level sensorthreshold   100.000 Measurement duration Background measurement 10.00 sduration (red) Sample measurement 10.00 s duration (red) Perform bluelight Yes measurement? Background measurement 10.00 s duration (blue)Sample measurement 10.00 s duration (blue) Assess light background Nostability Measurement sequence Aliquots 1 Automatic number of Nomeasurements Pre-alignment delay 0.00 s Number of measurements 5 Delaybetween measurements 0.00 s Pre-measurement delay 0.00 s Closemeasurement window No after measurement Measurement obscuration settingsAuto start measurement No Obscuration low limit 0.10% Obscuration highlimit 20.00%  Enable obscuration No filtering Measurement alarms UseBackground Check No Background Check Limits [1, 200], [20, 60] Accessorycontrol settings Accessory name Hydro MV Is accessory dry? No Stirrerspeed 2400 rpm Ultrasound percentage   0% Fill Dispersant Source AutoIdentifier Manual tank fill? No Degas after tank and cell fill YesSonicate to stability? No Ultrasound mode Pre- Measurement Degas afterpre-measurement No ultrasound Align after pre-measurement No ultrasoundUltrasonication duration 10.00 s Clean sequence settings Clean sequencetype Normal Sonicate during clean? Yes Manually Fill Tank During NoClean? Clean Dispersant Source Auto Identifier Clean Dispersant Level 0Sensor Threshold Degas After Clean? No Analysis settings Analysis modelGeneral Purpose Single result mode No Number of killed inner 0 detectorsBlue light detectors killed No Fine powder mode No Analysis sensitivityNormal Analysed as Mastersizer No 3000E? Result Settings Result range islimited Yes Low size 0.020 μm High Size 2000.000 μm Result Units VolumeExtend Result No Result Emulation No Viscosity A 500 mL sample wastransferred into a 600 mL container. Viscosity was then measured on aBrookfield RV Viscometer using Spindle No. 3 at 50 rpm at 4° C. After 30seconds of rotation, the reading was taken. Probiotic Microbiologicalenumeration according to the Viability - Food Chemical Codex (FCC) 10method (Food Live and Active Chemical Codex FCC10 First Supplement.Microbiological Monograph/Bacillus coagulans GBI-30,6086/ Counts. 3781.Published by United States Pharmacopeia., 2017). Taste A taste assessorwas served a gold standard sample deemed to have the appropriate levelof sweetness, sourness and taste profile. The taste assessor was thenserved a sample and compared the sample to the gold standard sample todetermine whether the sample has acceptable levels of sweet and sour,and without off flavors. Specific Gravity Mettler Toledo DA100 DensityMeter was used to determine the specific gravity/density of a sample

TABLE 3A Results of analyses listed in Table 2. SAMPLE SAMPLE SAMPLESAMPLE SAMPLE SAMPLE SAMPLE SAMPLE 1 2 3 4 5 6 7 8 Water Activity0.6-0.7 0.6-0.7 0.6-0.7 0.6-0.7 0.6-0.7 0.6-0.7 0.6-0.7 0.6-0.7 pH4.0-8.0 4.0-8.0 4.0-8.0 4.0-8.0 4.0-8.0 4.0-8.0 4.0-8.0 4.0-8.0Sedimentation 10% 10% 10% 20% 10% 10% 10% 10% Phase Separation N/A N/AN/A N/A N/A N/A N/A N/A Particle Size    1-1,000    1-1,000    1-1,000   1-1,000    1-1,000    1-1,000    1-1,000    1-1,000 (μm) Viscosity(cP) 1,000- 1,000- 1,000-  1-100   100-10,000   100-10,000    1-1,000   1-1,000 100,000 100,000 100,000 Probiotic 1M-1B 1M-1B 500M-10B500M-10B  500M-10B  500M-10B  500M-10B  500M-10B  Viability - Live andActive Microbiological Counts. (CFU/g by weight of the composition)Taste Conforms Conforms Conforms Conforms Conforms Conforms ConformsConforms Specific Gravity 1.0-1.5 1.0-1.5 1.0-1.5 1.0-1.5 1.0-1.51.0-1.5 1.0-1.5 1.0-1.5

TABLE 3B Results of analyses listed in Table 2. SAMPLE SAMPLE SAMPLESAMPLE SAMPLE SAMPLE SAMPLE SAMPLE 9 10 11 12 13 14 15 16 Water Activity0.6-0.7 0.6-0.7 0.6818 0.6-0.7 0.6-0.7 0.6-0.7 0.6-0.7 0.6-0.7 pH4.0-8.0 <4.6 4.27 <4.6 <4.6 <4.6 <4.6 <4.6 Sedimentation 10% N/A N/A N/AN/A N/A N/A N/A Phase Separation N/A N/A N/A N/A N/A 50% 50% N/AParticle Size    1-1,000    1-1,000 8.346    1-1,000    1-1,000   1-1,000    1-1,000    1-1,000 (μm) Viscosity (cP)    1-1,000   1-1,000 992    1-1,000    1-1,000   100-10,000   100-10,000  100-10,000 Probiotic 500M-10B  500M-10B  4.35B 500M-10B  500M-10B 500M-10B  500M-10B  500M-10B  Viability - Live and Active Micro. (CFU/gby weight of the composition) Taste Conforms Conforms Conforms ConformsConforms Conforms Conforms Conforms Specific Gravity 1.0-1.5 1.0-1.51.33 1.0-1.5 1.0-1.5 1.0-1.5 1.0-1.5 1.0-1.5

Example 3: Comparative Testing—Sedimentation

The liquid compositions were stored for two weeks without agitation. Theliquid compositions were visually inspected, and it was found that thecomponents of the liquid compositions did not sediment.

Example 4: Comparative Testing—Water Activity and Viscosity

The Enterogermina samples (US 2015/0118203) were tested for wateractivity and viscosity and compared to the samples herein. The Aw of theEnterogermina product was equal to 1. In contrast, the Aw of Samples1-16 herein is between about 0.6 to about 0.7.

Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the presentdisclosure. Unless specifically defined otherwise, all technical andscientific terms used herein shall be taken to have the same meaning ascommonly understood by one of ordinary skill in the art (e.g., in cellculture, bacteriology, molecular genetics, and biochemistry).

Values or ranges may be expressed herein as “about” and/or from/of“about” one particular value to another particular value. When suchvalues or ranges are expressed, other embodiments disclosed include thespecific value recited and/or from/of the one particular value toanother particular value. Similarly, when values are expressed asapproximations, by the use of antecedent “about,” it will be understoodthat here are a number of values disclosed therein, and that theparticular value forms another embodiment. It will be further understoodthat there are a number of values disclosed therein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. In embodiments, “about” can be used to mean, forexample, within 10% of the recited value, within 5% of the recited valueor within 2% of the recited value.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

As used in this specification and the appended claims, the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, in reference to defined or described elements of anitem, composition, apparatus, method, process, system, etc. are meant tobe inclusive or open ended, permitting additional elements, therebyindicating that the defined or described item, composition, apparatus,method, process, system, etc. includes those specified elements—or, asappropriate, equivalents thereof—and that other elements can be includedand still fall within the scope/definition of the defined item,composition, apparatus, method, process, system, etc.

For purposes of describing and defining the present teachings, it isnoted that unless indicated otherwise, the term “substantially” isutilized herein to represent the inherent degree of uncertainty that maybe attributed to any quantitative comparison, value, measurement, orother representation. The term “substantially” is also utilized hereinto represent the degree by which a quantitative representation may varyfrom a stated reference without resulting in a change in the basicfunction of the subject matter at issue.

As used herein, the term “germination-permissive conditions” areenvironmental conditions which allow for the germination of the spores.These environmental conditions include temperature, nutrients, pH levelsand the like.

As used herein, “shelf-stable” refers to a composition that is stableunder ambient, refrigerated or frozen conditions and safe forconsumption.

As used herein an “alimentary additive” is a solid and/or fluid nutrientadditive intended for human or animal consumption.

No limit is placed on the manner in which the shelf stablespore-containing probiotic alimentary additive described herein ismarketed, sold, distributed, stored, etc. For example, the examplesabove describe embodiments where the spore-containing probioticcompositions are packaged in various formats, such as in a bag with orwithout a cap and in a bag-in-box. The shelf stable spore-containingprobiotic alimentary additive can be distributed in these or similarformats for industrial processing for food and beverage applications.The shelf stable spore-containing probiotic alimentary additive can alsobe packaged and distributed for individual consumer consumption, such asfor a dietary supplement. This may include any well-known single servingand multi-serving packaging, including sealed vials, liquid supplements,etc.

While the above description may have focused on the use of aspore-forming probiotic bacterial strain and on conditions formaintaining dormancy, it will be understood that the shelf stablespore-containing probiotic alimentary additive could also be preparedusing (separate to the spores or in addition to the spores) encapsulated(non-sporulating) vegetative probiotics, such as, for example, those ofLactobacillus and Bifidobacterium species.

One skilled in the art will appreciate further features and advantagesof the present disclosure based on the above-described embodiments.Accordingly, the invention is not to be limited by what has beenparticularly shown and described, except as indicated by the appendedclaims. All publications and references cited herein are expresslyincorporated herein by reference in their entirety. Any patent,publication, or information, in whole or in part, that is said to beincorporated by reference herein is incorporated herein only to theextent that the incorporated material does not conflict with existingdefinitions, statements, or other disclosure material set forth in thisdocument. As such the disclosure as explicitly set forth hereinsupersedes any conflicting material incorporated herein by reference.

What is claimed is:
 1. A spore-containing probiotic composition,comprising: a pasteurized liquid slurry comprising, a probiotic materialcomprising spores from at least one spore-forming probiotic bacterialstrain wherein at least 90% of the spores are viable at pasteurizationtimes and temperatures; a saccharide, and at least one humectant,wherein the composition has a water activity (Aw) of less than about0.7, and a pH of about 8.0 or less.
 2. The composition of claim 1,wherein the at least one humectant is at least one of glycerin, a sugar,a salt, propylene glycol, or a gum.
 3. The composition of claim 1,wherein the at least one spore-forming probiotic bacterial straincomprises at least one Bacillus bacterial strain.
 4. The composition ofclaim 3, wherein the at least one spore-forming probiotic bacterialstrain comprises at least one of Bacillus coagulans bacterial strain, atleast one of Bacillus subtilis bacterial strain, at least one ofBacillus clausii bacterial strain, at least one of Bacillus indicusbacterial strain, at least one of Bacillus licheniformis bacterialstrain, at least one of Bacillus pumilus bacterial strain, at least oneof Bacillus amyloliquifaciens bacterial strain, or at least one ofBacillus megaterium bacterial strain.
 5. The composition of claim 3,wherein the at least one Bacillus coagulans bacterial strain comprisesBacillus coagulans GBI-30 strain (ATCC Designation Number PTA-6086). 6.The composition of claim 1, wherein the plurality of spores is presentin the composition in an amount from about 500 million CFU/g to 1trillion CFU/g by weight of the composition.
 7. The composition of claim1, further comprising at least one rheology modifier that does notcontain inulin, wherein the at least one rheology modifier is present inan amount from about 0.01% to 0.3% by weight of the composition.
 8. Thecomposition of claim 7, wherein the at least one rheology modifier is atleast one of xanthan gum, gum arabic, gellan gum, guar gum, orcarrageenan.
 9. A food product, comprising: a food, and thespore-containing probiotic composition of claim
 1. 10. A beverageproduct, comprising: a liquid beverage, and the spore-containingprobiotic composition of claim
 1. 11. A spore-containing probioticcomposition, comprising: a liquid slurry comprising, a probioticmaterial comprising spores from Bacillus coagulans GBI-30 strain (ATCCDesignation Number PTA-6086), wherein at least 90% of the spores areviable at pasteurization times and temperatures; a saccharide, at leastone humectant, wherein the composition has a water activity (Aw) of lessthan about 0.7, and a pH of about 8.0 or less.
 12. The composition ofclaim 1, wherein the at least one humectant is glycerin and the at leastone rheology modifier is xanthan gum, gellan gum or a combinationthereof.
 13. The composition of claim 12, wherein glycerin is present inan amount of from 43% to 55% by weight of the composition, and whereinxanthan gum, gellan gum or a combination thereof is present in an amountof from 0.01% to 0.10% by weight of the composition.
 14. The compositionof claim 1, wherein the pH of the composition is about 2.0 to about 8.0.15. The composition of claim 11, wherein the pH of the composition isabout 2.0 to about 8.0.
 16. The composition of claim 1, wherein the pHof the composition is less than about 5.0.
 17. The composition of claim11, wherein the pH of the composition is less than about 5.0.